Anti-Enteropeptidase Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for enteropeptidase on Western blot?

Full-length enteropeptidase (TMPRSS15) runs at approximately 115-140 kDa on Western blot, though the exact mobility depends on glycosylation state. The protein comprises 1019 amino acids with a calculated mass near 115 kDa, but extensive N-glycosylation shifts the apparent molecular weight higher. You may also detect proteolytic fragments, particularly a C-terminal serine protease domain around 35-50 kDa if endogenous processing has occurred in your sample. Duodenal lysates often show multiple bands reflecting post-translational processing. Positive control lysates from duodenum or jejunum tissue clarify the expected pattern.

Which human tissues express enteropeptidase at detectable levels for Western blot?

Enteropeptidase expression is highly restricted to the brush-border enterocytes of the duodenum and proximal jejunum. For Western blot, duodenal mucosal scrapings or biopsy lysates serve as strong positive controls. Expression drops sharply in distal small intestine and is absent in colon, stomach, and non-gastrointestinal tissues under physiological conditions. Some pancreatic ductal cell lines or duodenal adenocarcinoma lines may express low levels. If working with cell lines, confirm expression by RT-PCR before committing to Western blot, because most immortalized intestinal lines lose enteropeptidase expression in culture.

What is the recommended starting dilution for this enteropeptidase antibody in Western blot?

Start at 1:1000 dilution in blocking buffer (five percent non-fat milk or BSA in TBST) for overnight incubation at four degrees Celsius. This rabbit polyclonal is supplied at 100 micrograms and has been validated at 1:1000 for Western blot on human samples. If signal is weak with low-abundance samples, you can titre up to 1:500. For abundant duodenal lysates, 1:2000 may suffice and reduce background. Always include a no-primary-antibody control lane to assess non-specific binding of the secondary antibody, particularly important with tissue lysates rich in endogenous immunoglobulin.

Does this enteropeptidase antibody cross-react with mouse or rat tissue?

This antibody is validated for human enteropeptidase. Cross-reactivity with mouse or rat is not validated, though sequence homology in the protease domain suggests potential cross-reactivity. Mouse and rat enteropeptidase share approximately 60-70 percent identity with human orthologs, concentrated in the catalytic domain. If you plan to use rodent duodenal samples, expect that the antibody may recognize the target, but you should empirically confirm specificity by running parallel lanes with and without competitor peptide or by siRNA knockdown. We recommend titrating dilution and optimizing blocking conditions if working outside validated species.

Can I use this antibody for immunohistochemistry on paraffin-embedded duodenal biopsies?

Triple Point Biologics antibodies are validated for Western blot as a brand standard, so this enteropeptidase antibody is suitable for immunohistochemistry. For paraffin-embedded duodenal tissue, antigen retrieval is typically required—citrate buffer pH six at sub-boiling temperature for ten to twenty minutes works well for most serine protease epitopes. Enteropeptidase localizes to the apical brush border of duodenal enterocytes, so expect strong luminal-surface staining in well-oriented sections. Start at 1:200 to 1:500 dilution and include adjacent non-duodenal small intestine as a negative regional control.

How should I prepare duodenal tissue lysates to preserve enteropeptidase for Western blot?

Enteropeptidase is a serine protease susceptible to autoproteolysis and cleavage by trypsin once activated. Snap-freeze duodenal mucosal scrapings in liquid nitrogen immediately after harvest and store at minus eighty degrees Celsius. Lyse in RIPA or NP-40 buffer supplemented with protease inhibitor cocktail containing serine protease inhibitors such as PMSF or aprotinin. Keep samples on ice during lysis and clarify lysates promptly by centrifugation at four degrees Celsius. Avoid multiple freeze-thaw cycles, which promote fragmentation. Boil samples in Laemmli buffer immediately before loading to denature residual protease activity.

What is the proper storage condition for this rabbit polyclonal enteropeptidase antibody?

Store the antibody at minus twenty degrees Celsius in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The antibody is supplied as 100 micrograms in a buffered solution, typically containing glycerol or BSA as stabilizers. For routine use, keep a working aliquot at four degrees Celsius for up to one month; supplementing with zero point zero two percent sodium azide can extend working-stock stability if your downstream application permits. Do not store diluted antibody in blocking buffer for more than one week at four degrees, as proteolytic degradation and microbial contamination compromise performance.

Why do I see multiple bands on Western blot with this enteropeptidase antibody?

Enteropeptidase undergoes complex proteolytic maturation and can be cleaved into heavy- and light-chain fragments linked by disulfide bonds, similar to other type II serine proteases. Under reducing conditions you may resolve a light chain around thirty-five to fifty kilodaltons (the catalytic domain) and heavier N-terminal fragments. Glycosylation heterogeneity also broadens bands. If you see lower-molecular-weight species, consider whether your sample contains active trypsin or other pancreatic proteases that cleave enteropeptidase post-lysis; this is common in duodenal lysates without rigorous protease inhibition. Running a non-reducing gel alongside can help distinguish disulfide-linked complexes from degradation products.